The present invention relates to ligand-conjugated oligomeric compounds which bind to protein molecules and possess enhanced pharmacokinetic properties. The present invention further relates to methods for increasing the concentration of oligomeric compounds in serum and methods for promoting the cellular uptake of oligomeric compounds in cells.
Protein synthesis is directed by nucleic acids through the intermediacy of messenger RNA (mRNA). Antisense methodology is the complementary hybridization of relatively short oligonucleotides to mRNA or DNA such that the normal, essential functions, such as protein synthesis, of these intracellular nucleic acids are disrupted. Hybridization is the sequence-specific hydrogen bonding via Watson-Crick base pairs of oligonucleotides to RNA or single-stranded DNA. Such base pairs are said to be complementary to one another.
The naturally-occurring events that provide the disruption of the nucleic acid function, discussed by Cohen (Oligonucleotides: Antisense Inhibitors of Gene Expression, CRC Press, Inc., 1989, Boca Raton, Fla.) are thought to be of two types. The first, hybridization arrest, describes the terminating event in which the oligonucleotide inhibitor binds to the target nucleic acid and thus prevents, by simple steric hindrance, the binding of essential proteins, most often ribosomes, to the nucleic acid. Methyl phosphonate oligonucleotides (Miller et al. (1987) Anti-Cancer Drug Design, 2:117-128), and xcex1-anomer oligonucleotides are the two most extensively studied antisense agents which are thought to disrupt nucleic acid function by hybridization arrest.
Another means by which antisense oligonucleotides disrupt nucleic acid function is by hybridization to a target mRNA, followed by enzymatic cleavage of the targeted RNA by intracellular RNase H. A 2xe2x80x2-deoxyribofuranosyl oligonucleotide or oligonucleotide analog hybridizes with the targeted RNA and this duplex activates the RNase H enzyme to cleave the RNA strand, thus destroying the normal function of the RNA. Phosphorothioate oligonucleotides are the most prominent example of an antisense agent that operates by this type of antisense terminating event.
Considerable research is being directed to the application of oligonucleotides and oligonucleotide analogs as antisense agents for diagnostics, research applications and potential therapeutic purposes. One of the major hurdles that has only partially been overcome in vivo is efficient cellular uptake which is severely hampered by the rapid degradation and excretion of oligonucleotides. The generally accepted process of cellular uptake is by receptor-mediated endocytosis which is dependent on the temperature and concentration of the oligonucleotides in serum and extra vascular fluids.
Efforts aimed at improving the transmembrane delivery of nucleic acids and oligonucleotides have utilized protein carriers, antibody carriers, liposomal delivery systems, electroporation, direct injection, cell fusion, viral vectors, and calcium phosphate-mediated transformation. However, many of these techniques are limited by the types of cells in which transmembrane transport is enabled and by the conditions needed for achieving such transport. An alternative that is particularly attractive for the transmembrane delivery of oligonucleotides is modification of the physicochemical properties of oligonucleotides via conjugation to a molecule that facilitates transport. Another alternative is to increase the stability of oligonucleotides in serum, thereby increasing their concentration and distribution.
It has been previously reported that oligonucleotides modified with a 4-[(N-2-chloroethyl-N-methyl)amino]benzylamine reactive functionality at a 5xe2x80x2-phosphate position react with albumin and immunoglobulins M and G (Yu et al., FEBS Letters, 1994, 334:96-98). Binding to albumin was weak at about 20 xcexcM with immunoglobulin binding stronger at about 4 to 6 xcexcM. This study further reported that oligonucleotides conjugated to steroids had increased affinity for blood cells and thus changed their distribution and increased their lifetime in serum.
One method for increasing membrane or cellular transport of oligonucleotides is the attachment of a pendant lipophilic group. Ramirez et al. (J. Am. Chem. Soc., 1982, 104:5483) introduced the phospholipid group 5xe2x80x2-O-(1,2-di-O-myristoyl-sn-glycero-3-phosphoryl) into the dimer TpT independently at the 3xe2x80x2 and 5xe2x80x2 positions. Subsequently Shea et al. (Nuc. Acids Res., 1990, 18:3777) disclosed oligonucleotides having a 1,2-di-O-hexyldecyl-rac-glycerol group linked to a 5xe2x80x2-phosphate on the 5xe2x80x2-terminus of the oligonucleotide. Certain of the Shea et al. authors also disclosed these and other compounds in patent application PCT/US90/01002. A further glucosyl phospholipid was disclosed by Guerra et al., Tetrahedron Letters, 1987, 28:3581.
In other work, a cholesteryl group was attached to the internucleotide linkage between the first and second nucleotides (from the 3xe2x80x2 terminus) of an oligonucleotide. This work is disclosed in U.S. Pat. No. 4,958,013 and further in Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553. Additional approaches to the delivery and study of oligonucleotides have involved the conjugation of a variety of other molecules and reporter groups. The aromatic intercalating agent anthraquinone was attached to the 2xe2x80x2 position of a sugar fragment of an oligonucleotide as reported by Yamana et al. (Bioconjugate Chem., 1990, 1:319), Lemairte et al. (Proc. Natl. Acad. Sci. USA, 1986, 84:648) and Leonetti et al. (Bioconjugate Chem., 1990, 1:149).
Lysine and polylysines have also been conjugated to oligonucleotides to improve their charge-size characteristics. The poly(L-lysine) was linked to the oligonucleotide via periodate oxidation of the 3xe2x80x2-terminal ribose followed by reduction and coupling through a N-morpholine ring. Oligonucleotide-poly(L-lysine) conjugates are described in European Patent application 87109348.0. In this instance, the lysine residue was coupled to a 5xe2x80x2 or 3xe2x80x2 phosphate of the 5xe2x80x2 or 3xe2x80x2 terminal nucleotide of the oligonucleotide. A disulfide linkage has also been utilized at the 3xe2x80x2 terminus of an oligonucleotide to link a peptide to the oligonucleotide. See, Corey and Schultz, Science, 1987, 238:1401; Zuckermann et al., J. Am. Chem. Soc., 1988, 110:1614; and Corey et al., J. Am. Chem. Soc., 1989, 111:8524.
A linking reagent for attaching biotin to the 3xe2x80x2-terminus of an oligonucleotide has also been described. Nelson et al., Nuc. Acids Res., 1989, 17:7187. This reagent, N-Fmoc-O-DMT-3-amino-1,2-propanediol is now commercially available from Clontech Laboratories (Palo Alto, Calif.) under the name 3xe2x80x2-Amine on. It is also commercially available under the name 3xe2x80x2-Amino-Modifier reagent from Glen Research Corporation (Sterling, Va.). This reagent was also utilized to link a peptide to an oligonucleotide as reported by Judy et al. (Tetrahedron Letters, 1991, 32:879). A similar commercial reagent (actually a series of such linkers having various lengths of polymethylene connectors) for linking to the 5xe2x80x2-terminus of an oligonucleotide is 5xe2x80x2-Amino-Modifier C6. These reagents are available from Glen Research Corporation (Sterling, Va.). These compounds or similar ones were utilized by Krieg et al. (Antisense Research and Development, 1991, 1:161) to link fluorescein to the 5xe2x80x2-terminus of an oligonucleotide. Other compounds of interest have also been linked to the 3xe2x80x2-terminus of an oligonucleotide. Asseline et al. (Proc. Natl. Acad. Sci. USA, 1984, 81:3297) describe linking acridine on the 3xe2x80x2-terminal phosphate group of an poly (Tp) oligonucleotide via a polymethylene linkage. Haralambidis et al. (Tetrahedron Letters, 1987, 28:5199) report building a peptide on a solid state support and then linking an oligonucleotide to that peptide via the 3xe2x80x2 hydroxyl group of the 3xe2x80x2 terminal nucleotide of the oligonucleotide. Chollet (Nucleosides and Nucleotides, 1990, 9:957) attached an Aminolink 2 (Applied Biosystems, Foster City, Calif.) to the 5xe2x80x2 terminal phosphate of an oligonucleotide. The bifunctional linking group SMPB (Pierce Chemical Co., Rockford, Ill.) was then used to link an interleukin protein to the oligonucleotide.
Conjugation of lipids, reporters, peptides and other molecules to oligonucleotides is not limited to the terminal 3xe2x80x2 and 5xe2x80x2-positions. A wide variety of conjugates have also been reported in the literature wherein attachment is performed at any one or more of the 2xe2x80x2-positions on the nucleotide building blocks of the oligonucleotide. Further conjugates have also been reported wherein attachment occurs on the internucleotide linkage or on one of the atoms of the nucleobase of any one of the nucleotide units of the oligonucleotide. For example, an EDTA iron complex has been linked to the 5 position of a pyrimidine nucleoside as reported by Dreyer and Dervan (Proc. Natl. Acad. Sci. USA, 1985, 82:968). Fluorescein has been linked to an oligonucleotide in the same manner as reported by Haralambidis et al. (Nucleic Acid Research, 1987, 15:4857) and biotin in the same manner as described in PCT application PCT/US/02198. Fluorescein, biotin and pyrene were also linked in the same manner as reported by Telser et al. (J. Am. Chem. Soc., 1989, 111:6966). A commercial reagent, Amino-Modifier-dT, from Glen Research Corporation (Sterling, Va.) can be utilized to introduce pyrimidine nucleotides bearing similar linking groups into oligonucleotides.
Manoharan et al. (PCT Application WO 93/07883) have also reported the conjugation of oligonucleotides with a variety of molecules such as steroids, reporter molecules, reporter enzymes, vitamins, non-aromatic lipophilic molecules, chelators, porphyrins, intercalators, peptides and proteins through the intermediacy of varied linking groups, such as 6-aminoalkoxy and 6-aminoalkylamino groups. Conjugation has been reported at the 3xe2x80x2-, 5xe2x80x2-, 2xe2x80x2-, internucleotide linkage and nucleobase positions of oligonucleotides. Such oligonucleotide conjugates are expected to have improved physicochemical properties that facilitated their uptake and delivery into cells as demonstrated by in vitro experiments. The intracellular and intranuclear delivery of nucleic acids and oligonucleotides, however, is still a challenge. Most often, penetration of heretofore reported oligonucleotide conjugates has been found to be limited. This has typically been a problem because such conjugates have generally been designed to improve the passive absorption of the oligonucleotides where the size, physicochemical properties and extracellular concentration of the conjugate play important limiting roles. This coupled with the limited extracellular stability of nucleic acids and oligonucleotides demands the development of novel conjugates that will deliver higher levels of nucleic acids and oligonucleotides into specific tissues and targeted cells.
Albumin is the most abundant protein in mammalian systems, and plays an important role in the transport and deposition of drug substances in blood. It is generally accepted that there are two major specific drug binding sites, site I and site II on human albumin. X-ray studies of crystalline human albumin (He and Carter, Nature, 1992, 358:209-215) indicate that site I and site II are located within specialized cavities in subdomain IIA and IIIA, respectively.
Interaction of oligonucleotides with proteins play an important role in absorption, distribution and pharmacokinetics. In the bloodstream, the major oligonucleotide binding protiens are immunoglobulins M and G, serum albumin, and orosomucoid xcex1-1-acid glycoprotein (AAG). The role of plasma protein binding is an important factor in oligonucleotide disposition and efficacy. If protein binding of oligonucleotides can be modulated with small molecular conjugation, it will result in more efficacious oligonucleotide drugs.
Albumin is a water-soluble protein with a molecular eight of 66,500 comprising a single chain of 585 amino acids containing a single tryptophan (Trp-214), low (2%) glycine content, high cystine content and a large number of charged amino acids (about 100 negative charges and 100 positive charges) and has an isoelectric point of about pH 5.0. Thus, at a plasma pH of 7.4, it has a net negative charge of xe2x88x9215. Nonetheless, it attracts both anions and cations. It circulates at a concentration of 3.5-5 g/100 mL in blood plasma and also exists at lower concentrations in extravascular fluids. About 60% of all human serum albumin (HSA) is located in the extravascular space (Peters, Adv. Protein Chemn., 1985, 37:161). As the most abundant protein in plasma, HSA plays an important role in the maintenance of blood pH and colloidal osmotic pressure and accounts for most of the thiol content of plasma (Cys-34). Binding of drugs to albumin is usually rapidly reversible. The binding (association) constants are typically in the range of 104 to 106 Mxe2x88x921. HSA is organized in a series of three repeating domains (I, II and III) each having two subdomains. Ligands bind to HSA generally to one or both of two binding sites. Site I is associated with the ligands warfarin, phenyl butazone. This site is localized in subdomain IIA. Site II is in subdomain IIIA and binds to diazepam and ibuprofen. Other ibuprofen analogs suprofen, pranoprofen, carprofen, fenbufen and ketoprofen, which are all non-steroidal antiinflammatory agents bind to site II. Flufenamic acid and dansylsarcosine bind to site II while dansylamide bind to site I. Barbiturates such as quinalbarbitone interact with site II and the antidiabetic tolbutamide binds to site I, site II and an unidentified site. (R)-Folinic acid binds to both sites. Other compounds that bind to HSA include thiadiazides, diazepines, and antibacterials (e.g., nalidixic acid).
Lipoproteins can contribute to the plasma binding of lipophilic drugs and dissolve in lipid core of the lipoproteins. Cholesterol conjugated oligonucleotides are known to bind to serum proteins. Agrawal et al., (xe2x80x9cEffect of aspirin on protein binding and tissue disposition of oligonucleotide phosphorothioate in rats,xe2x80x9d Journal of Drug Targeting, 1998, 5:303-313) describe the effect of co-administration of aspirin at a concentration of 2 mg/mL and demonstrate that the Pxe2x95x90S oligonucleotide binding to serum albumin is reduced (as measured by % protein bound of Pxe2x95x90S oligonucleotide). This result indicates that presence of aspirin in the body or similar small molecule drugs could effectively alter protein binding of Pxe2x95x90S oligonucleotides in vivo.
Pharmacokinetic studies of Pxe2x95x90S oligonucleotide (GEM-91, 25-mer phosphorothioate oligonucleotide) in rats were determined after bolus injection. One hour before administration of the drug, aspirin is administered by gavage. When Pxe2x95x90S oligonucleotide was administered following aspirin administration in rats the following the plasma pharmacokinetic parameters (txc2xd xcex1, txc2xd xcex2, AUC, etc.) were lower. The tissue disposition was significantly different in that the majority of tissues. e.g. kidney, liver, spleen, bone marrow, skin, thyroid, adrenal, heart, lung, and pancreas, had lower concentrations, and gastrointestinal tissues and contents had a higher concentration. In certain tissues, e.g. liver and bone marrow, the concentration of Pxe2x95x90S oligonucleotide which was administered following aspirin administration was about half of that observed following administration of Pxe2x95x90S oligonucleotide alone. It was seen that the rate of elimination was affected in animals compared to rats receiving Pxe2x95x90S oligonucleotide alone. A higher concentration of excreted oligonucleotide in feces from rats receiving Pxe2x95x90S oligonucleotide following aspirin was observed compared to rats receiving Pxe2x95x90S oligonucleotide alone. However, the effect of attaching small molecule drugs to the oligonucleotide to modulate serum albumin binding has not been studied.
Therefore, there is a clear need for oligonucleotide conjugates having improved distribution and cellular uptake and methods for their preparation, that address the shortcomings of oligonucleotide conjugates as described above. The present invention is directed to this very important end.
The present invention provides ligand conjugated oligomeric compounds that are capable of interacting with a protein. In particular, the ligand conjugated oligomeric compounds of the present invention bind to proteins. More particularly, the present invention provides oligomeric compounds that are conjugated to drug moieties.
The oligomeric compounds of the present invention bind to serum, vascular and cellular proteins. It is preferred that the serum proteins include albumin, an immunoglobulin, a lipoprotein, xcex1-2-macroglobulin and xcex1-1-glycoprotein.
The present invention also provides ligand conjugated oligomeric compounds wherein the oligomeric compound is an oligonucleotide comprising a plurality of nucleosides. Also provided are oligonucleotides wherein the nucleosides are connected by phosphodiester linkages. Further, oligonucleotides wherein the nucleosides are connected by phosphorothioate linkages are also provided. It is preferred that at least one of the nucleosides of the oligonucleotides of the present invention bear a 2xe2x80x2-substituent group.
The present invention also provides methods for increasing the concentration of an oligonucleotide in serum comprising the steps of:
(a) selecting a drug moiety that is known to bind to a serum protein;
(b) conjugating said drug moiety to said oligonucleotide to form a conjugated oligonucleotide; and
(c) adding said conjugated oligonucleotide to said serum.
The present invention further provides methods for increasing the capacity of serum for an oligonucleotide comprising the steps of:
(a) selecting a drug moiety that is known to bind to a serum protein;
(b) conjugating said drug moiety to said oligonucleotide to form a conjugated oligonucleotide; and
(c) adding said conjugated oligonucleotide to said serum.
In one embodiment of the present invention the serum protein is a protein having a binding site for the drug moiety. In another embodiment the serum protein is a protein having a binding site for the oligonucleotide. In yet another embodiment the serum protein is a protein having a binding site for the oligonucleotide and a binding site for the drug moiety such that the binding site for the oligonucleotide is distinct from the binding site for the drug moiety.
The present invention further provides methods for increasing the binding of an oligonucleotide to a portion of the vascular system comprising the steps of:
(a) selecting a drug moiety that is known to bind to a protein that resides, in part, in the circulating serum and, in part, in a non-circulating portion of the vascular system;
(b) conjugating said drug moiety to said oligonucleotide to form a conjugated oligonucleotide; and
(c) adding said conjugated oligonucleotide to said vascular system.
The present invention also provides methods for promoting cellular uptake of an oligonucleotide in a cell comprising the steps of:
(a) selecting a protein that resides on the cellular membrane and extends, at least in part, on the external side of said membrane;
(b) selecting a drug moiety that is known to bind to said protein;
(c) conjugating said drug moiety to said oligonucleotide to form a conjugated oligonucleotide; and
(d) exposing said cell to said conjugated oligonucleotide.
Preferably, the protein residing on the cellular membrane is a cell surface integrin.
In one embodiment of the present invention the serum protein is albumin, an immunoglobulin, xcex1-2-macroglobulin, xcex1-1-glycoprotein or a lipoprotein. Preferably, the serum protein is albumin.
In yet another embodiment of the present invention the drug moiety is aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic. Preferably, the drug moiety is aspirin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, palmityl or carprofen. More preferably, the drug moiety is ibuprofen.